1. Field of the Invention
The present invention relates to an illumination apparatus for a microscope.
2. Description of the Related Art
In the research of analyzing the dynamics and functions of organization intracellular proteins and an organelle, an experiment is widely performed in which a specific intracellular portion is irradiated with light to observe a resultant reaction. This experiment uses a fluorescence sample obtained by labeling a fluorescent material, which causes a photoirradiation reaction, with a specific intracellular material as the observation target by antibody staining or gene injection. In accordance with the aspects of the photoirradiation reactions of various fluorescent materials, experiment schemes that enhance the features of the fluorescent materials have been proposed.
A typical example of the experiment scheme includes one (to be referred to as Caged experiment hereinafter) that uses a reagent called a Caged compound. The Caged compound is a material obtained by chemically modifying a physiologically active material by a protecting group to inactivate it. When the Caged compound is irradiated with ultraviolet light (having a center wavelength of approximately 360 nm), its protecting group dissociates to locally free a necessary physiologically active material. By using these characteristics, only a portion irradiated with the ultraviolet light can be activated. Thus, this scheme is widely used as a scheme that controls the location and time where the intracellular protein is to be activated.
Another typical example is an experiment (to be referred to as kaede experiment hereinafter) that uses a fluorescent protein called kaede (to be referred to as kaede protein hereinafter). The characteristic feature of the kaede protein is as follows. When the kaede protein is irradiated with light having a wavelength range of approximately 405 nm, the peak of the fluorescence wavelength changes from 518 nm (green) to 580 nm (red). When a gene of the kaede protein is injected into a desired intracellular protein and allowed to express, the portion irradiated with 405-nm light emits red fluorescence while the remaining portion emits green fluorescence. By using this characteristic feature, when a desired intracellular portion is irradiated with 405-nm light, only a protein that locally exists there can be discolored to red. Thus, how the red protein propagates in the cell can be observed. Also, the entire cell can be discolored to red to discriminate it from other cells.
In both the Caged experiment and kaede experiment, during the fluorescence observation, a desired portion is irradiated with ultraviolet light or 405-nm light (to be referred to as stimulus light hereinafter) at a desired timing. Then, how the dynamics of the fluorescence differ before and after the irradiation and how the protein propagates in the cell are observed. Therefore, to conduct these experiments, two types of illumination, i.e., local illumination and fluorescence observation illumination, are required. In the local illumination, stimulus light is locally applied to a desired position in the observation range of the sample. In the fluorescence observation illumination, excitation light is applied to the entire observation range. In particular, assume that intracellular protein diffusion or activating phenomenon occurs within a short period of time of several sec to 1 sec or less. In this case, fluorescence observation illumination must be able to be performed while performing local illumination simultaneously.
Conventionally, illumination apparatuses have been disclosed that applies two types of illumination to the sample simultaneously.
An illumination apparatus disclosed in Jpn. Pat. Appln. KOKAI Publication No. 07-056092 comprises independently a local illumination optical system 623 and a fluorescence observation illumination optical system 622, as shown in FIG. 6. The local illumination optical system 623 comprises a light source 641 and a local illumination stop 645. The fluorescence observation illumination optical system 622 comprises a light source 631 and a field stop 634. Local illumination light from the local illumination optical system 623 and fluorescence observation illumination light from the observation illumination optical system 622 are combined by a dichroic mirror 635. Thus, the two light beams are applied to a sample S simultaneously through a field stop projection lens 636, and a dichroic mirror 625 and an objective lens 624 of an observation optical system 621.
An illumination apparatus disclosed in Jpn. Pat. Appln. KOKAI Publication No. 10-090608 is configured as follows, as shown in FIG. 7. Excitation light C output from an excitation light source 732 is applied to a sample 750 through a condenser lens 733, a bandpass filter 734, dichroic mirrors 735 and 741, and an objective lens 742. A light beam projected from an irradiation light source 710 is split by a branching optical system 720. The split light beams are locally applied to the different positions of the sample 750 through a condenser lens 731, the dichroic mirrors 735 and 741, and the objective lens 742.
An illumination apparatus disclosed in Jpn. Pat. Appln. KOKAI Publication No. 2004-177662 is configured as follows, as shown in FIG. 8. A light beam of illumination light projected from a light source 811 is split into three light beams by half mirrors 821 and 881. The three split light beams are subjected to wavelength selection by excitation filters 824A, 824B, and 8240. The three light beams having the selected wavelengths are combined by half mirrors 882 and 825 into one light beam to be applied to a sample 843 via a dichroic mirror 841 and an objective lens 842.
When conducting Caged experiment or kaede experiment, it must be checked in advance whether the position of local illumination on the sample coincides with the position desired by the person in charge of the experiment. As soon as irradiation with stimulus light is started, the sample starts reaction. To check the position of local illumination in advance, local illumination must be performed using visible light having a wavelength range that does not cause reaction. This visible light will be referred to as guide light hereinafter. The guide light desirably has a long wavelength range (e.g., red light) that is separated as far as possible from the wavelength range of the stimulus light.